Auditory impairments and vestibular defects afflict approximately 30 million and 25 million Americans, respectively. A frequent causative factor is damage to the sensory hair cells of these systems. The hair cell mechanotransduction cascade converts the mechanical signals of sound and motion into electrical signals, which are transmitted to the nervous system. A lack of understanding of mechanotransduction at the molecular level hinders development of effective therapies for deafness and vestibular disorders. Calcium ions play a critical role in hair cell mechanotransduction. Mice lacking functional PMCA2 (plasma membrane Ca2+ATPase 2) are deaf with vestibular defects. This pathology is likely due to the lack of proper Ca2+ extrusion from hair cells, indicating that PMCA2 is fundamental for setting the resting Ca2+ level and maintaining the transmembrane Ca2+ gradient near the site of mechanotransduction. The goal of this application is to directly assess the role of PMCA2 in hair cell transduction. A mouse will be engineered to express a mutant PMCA2 that is sensitive to inhibition by thiol-reactive adenosine derivatives. The effects of PMCA2 inhibition on Ca2+ homeostasis and mechanotransduction will be assessed with Ca2+ imaging and electrophysiological approaches. [unreadable] [unreadable]